Study on the Antifungal Activity of Gallic Acid and Its Azole Derivatives against Fusarium graminearum

The wheat scab caused by Fusarium graminearum (F. graminearum) has seriously affected the yield and quality of wheat in China. In this study, gallic acid (GA), a natural polyphenol, was used to synthesize three azole-modified gallic acid derivatives (AGAs1–3). The antifungal activity of GA and its derivatives against F. graminearum was studied through mycelial growth rate experiments and field efficacy experiments. The results of the mycelial growth rate test showed that the EC50 of AGAs–2 was 0.49 mg/mL, and that of AGAs–3 was 0.42 mg/mL. The biological activity of AGAs–3 on F. graminearum is significantly better than that of GA. The results of field efficacy tests showed that AGAs–2 and AGAs–3 significantly reduced the incidence rate and disease index of wheat scab, and the control effect reached 68.86% and 72.11%, respectively. In addition, preliminary investigation was performed on the possible interaction between AGAs–3 and F. graminearum using density functional theory (DFT). These results indicate that compound AGAs–3, because of its characteristic of imidazolium salts, has potential for use as a green and environmentally friendly plant-derived antifungal agent for plant pathogenic fungi.


Introduction
Wheat is one of the important global food crops and is susceptible to various pests and pathogens.Fusarium graminearum (F.graminearum) is one of the most serious pathogens, causing a serious decrease in wheat yield and quality due to Fusarium head blight (FHB or wheat scab) [1,2].Moreover, the various secondary metabolites produced by F. graminearum during wheat infection can increase the risk of cancer in humans and animals [3].Chemical control is one of the main measures to prevent and control wheat scab.However, the pathogen of wheat scab has developed resistance to various pesticides because of the longterm and large-scale use of chemical drugs, leading to a decrease in control effectiveness and causing environmental pollution and food security issues [4].Therefore, finding efficient, broad-spectrum, low-cost, and environmentally friendly antifungal compounds is an urgent issue for technical personnel in this field.
Natural products, with advantages such as biocompatibility, structural diversity, and unique mechanisms of action, are considered an important source for developing new fungicides [5,6].Gallic acid (GA) is a natural polyphenolic compound found in various plants, such as chestnuts, gallnuts, sumac trees, witch hazels, watercress, oak bark, tea, betel nuts, and blackberries [7], with a widely and easily obtainable source.Various pharmaceutical and chemical industries have expressed interest in GA due to its biological activities and commercial applications such as antioxidant, anticancer, antibacterial, antiviral, antiinflammatory, etc. [8][9][10][11][12][13][14].Thus, due to its abundance in nature and diversity of bioactivity, GA is considered an important molecule with significant development prospects for designing new and effective drugs.In the past decades of their development, GA derivatives have shown various excellent biological activities [15,16].For example, the alkyl and arylgallates of GA exhibit high biological activity attributed to their long chains, which may help to improve cell permeability and lipid or hydrophobic pockets in target molecules [17].Other derivatives with various activities have been investigated one after another, such as linear and branched gallic acid containing an amide moiety, gallic acid hydrazides, galloyl-heterocyclic hybrids, peptide-based hybrids, sugar-based hybrids, etc. [10,[18][19][20][21][22].
Azoles are a large class of five-membered heterocycles containing one or more nitrogen atoms, with or without other heteroatoms, and have always been considered as the preferred scaffold for designing novel therapeutic drugs [23].Various nitrogen-containing and oxygen-containing azole compounds with aromatic and electron rich properties, such as imidazole, pyrazole, triazole, tetrazole, thiazole, etc., have been widely studied due to their diverse biological activities [24,25].Previous studies have shown that azole derivatives, especially for pyrazole and imidazole derivatives (structures shown in Figure 1), have various biological activities, such as antifungal, antibacterial, anticancer, anti-inflammatory, and antituberculosis activities [26][27][28][29][30][31][32][33][34][35].Xu et al. synthesized a series of aroyl pyrrolidine derivatives and investigated their in vitro inhibitory activity and in vivo anti-tumor activity against gelatinase (MMP-2, -9), of which most inhibitors exhibit good inhibitory activity (inhibition rate > 35%) [36].Moreover, acyl pyrazoline derivatives also exhibit significant inhibitory effects on LPS stimulated NO generation and can effectively clear DPPH free radicals [37].In addition, quaternary ammonium salts (QAS), with their unique structure, broad-spectrum antibacterial properties, low toxicity, good water solubility, and easy synthesis, are widely used in various fields such as agricultural fungicides, public place disinfection, and water treatment [38,39].
pharmaceutical and chemical industries have expressed interest in GA due to its biological activities and commercial applications such as antioxidant, anticancer, antibacterial, antiviral, anti-inflammatory, etc. [8][9][10][11][12][13][14].Thus, due to its abundance in nature and diversity of bioactivity, GA is considered an important molecule with significant development prospects for designing new and effective drugs.In the past decades of their development, GA derivatives have shown various excellent biological activities [15,16].For example, the alkyl and arylgallates of GA exhibit high biological activity a ributed to their long chains, which may help to improve cell permeability and lipid or hydrophobic pockets in target molecules [17].Other derivatives with various activities have been investigated one after another, such as linear and branched gallic acid containing an amide moiety, gallic acid hydrazides, galloyl-heterocyclic hybrids, peptide-based hybrids, sugar-based hybrids, etc. [10,[18][19][20][21][22].
Azoles are a large class of five-membered heterocycles containing one or more nitrogen atoms, with or without other heteroatoms, and have always been considered as the preferred scaffold for designing novel therapeutic drugs [23].Various nitrogencontaining and oxygen-containing azole compounds with aromatic and electron rich properties, such as imidazole, pyrazole, triazole, tetrazole, thiazole, etc., have been widely studied due to their diverse biological activities [24,25].Previous studies have shown that azole derivatives, especially for pyrazole and imidazole derivatives (structures shown in Figure 1), have various biological activities, such as antifungal, antibacterial, anticancer, anti-inflammatory, and antituberculosis activities [26][27][28][29][30][31][32][33][34][35].Xu et al. synthesized a series of aroyl pyrrolidine derivatives and investigated their in vitro inhibitory activity and in vivo anti-tumor activity against gelatinase (MMP-2, -9), of which most inhibitors exhibit good inhibitory activity (inhibition rate > 35%) [36].Moreover, acyl pyrazoline derivatives also exhibit significant inhibitory effects on LPS stimulated NO generation and can effectively clear DPPH free radicals [37].In addition, quaternary ammonium salts (QAS), with their unique structure, broad-spectrum antibacterial properties, low toxicity, good water solubility, and easy synthesis, are widely used in various fields such as agricultural fungicides, public place disinfection, and water treatment [38,39].Herein, taking into account the above aspects, this study used natural polyphenol GA as the lead compound, introduced imidazole and pyrazole into GA, and quaternized them to synthesize a series of azole-modified gallic acid derivatives (AGAs) to exert their synergistic biological activity.Then, the antifungal effects of these derivatives against the wheat Fusarium head blight pathogen, F. graminearum, was investigated both in vitro and in vivo, and the interaction between drugs and receptors was given a preliminary explanation through computational chemistry methods.

Structural Characterization of GA and Its Derivatives
GA was obtained from the discarded chestnut leaves.To synthesize AGAs1-3, the intermediate 3,4,5-trimethoxybenzoic acid (TMBA) needed to be synthesized.In this experiment, GA was used as the starting material to first synthesize TMBA, and then AGAs1-2 were synthesized through the one-pot synthesis method using Im and Py as modified functional groups, respectively, as shown in Figure 2. AGAs-3 was prepared by heating and refluxing AGAs-2 with benzyl chloride.The post-treatment of this reaction is simple, and the yield is almost 100%.The structures of compounds GA, TMBA, and AGAs1-3 were characterized by nuclear magnetic resonance spectroscopy (NMR) and the 1 H NMR and 13 C NMR signal peaks of the characteristic functional groups of compounds were analyzed and assigned.This can be a mutual corroboration with the results of highresolution mass spectrometry (HR-ESI(±)-MS).The NMR spectrum and HR-ESI(±)-MS are supplied in the supporting materials.Herein, taking into account the above aspects, this study used natural polyphenol GA as the lead compound, introduced imidazole and pyrazole into GA, and quaternized them to synthesize a series of azole-modified gallic acid derivatives (AGAs) to exert their synergistic biological activity.Then, the antifungal effects of these derivatives against the wheat Fusarium head blight pathogen, F. graminearum, was investigated both in vitro and in vivo, and the interaction between drugs and receptors was given a preliminary explanation through computational chemistry methods.

Structural Characterization of GA and Its Derivatives
GA was obtained from the discarded chestnut leaves.To synthesize AGAs1-3, the intermediate 3,4,5-trimethoxybenzoic acid (TMBA) needed to be synthesized.In this experiment, GA was used as the starting material to first synthesize TMBA, and then AGAs1-2 were synthesized through the one-pot synthesis method using Im and Py as modified functional groups, respectively, as shown in Figure 2. AGAs-3 was prepared by heating and refluxing AGAs-2 with benzyl chloride.The post-treatment of this reaction is simple, and the yield is almost 100%.The structures of compounds GA, TMBA, and AGAs1-3 were characterized by nuclear magnetic resonance spectroscopy (NMR) and the 1 H NMR and 13 C NMR signal peaks of the characteristic functional groups of compounds were analyzed and assigned.This can be a mutual corroboration with the results of highresolution mass spectrometry (HR-ESI(±)-MS).The NMR spectrum and HR-ESI(±)-MS are supplied in the supporting materials.

Inhibitory Effects of GA and Its Azole Derivatives on the Mycelial Growth Rate and Spore Germination of F. graminearum In Vitro
The inhibitory effects of five compounds (GA, TMBA, and AGAs1-3) on the mycelial growth of F. graminearum at different concentrations are shown in Figure 3A.The azolemodified GA compounds AGAs1-3 had a significant inhibitory effect on the mycelial growth of F. graminearum.The inhibitory rate of compounds AGAs1-3 on the mycelial

Inhibitory Effects of GA and Its Azole Derivatives on the Mycelial Growth Rate and Spore Germination of F. graminearum In Vitro
The inhibitory effects of five compounds (GA, TMBA, and AGAs1-3) on the mycelial growth of F. graminearum at different concentrations are shown in Figure 3A.The azolemodified GA compounds AGAs1-3 had a significant inhibitory effect on the mycelial growth of F. graminearum.The inhibitory rate of compounds AGAs1-3 on the mycelial growth of F. graminearum was 11.00-73.46% in the concentration range of 0.05-0.8mg/mL.Among the AGAs compounds, AGAs-3 had the highest inhibition rate on the growth of F. graminearum hyphae at a concentration of 0.8 mg/mL, reaching 73.46%.However, the inhibitory rates of compounds GA and TMBA on the mycelial growth of F. graminearum were both zero at concentrations of 0.05-0.4mg/mL.When the concentration was 0.8 mg/mL, the inhibitory rates on the mycelial growth of F. graminearum were 9.93% and 9.55%, respectively.It can be seen that at different concentrations, the inhibitory effect of compounds AGAs1-3 on the mycelial growth of F. graminearum is significantly better than that of unmodified compound GA, indicating that compared with the unmodified complex GA, the azole-modified GA derivatives AGAs1-3 significantly enhance its antifungal activity against F. graminearum due to the introduction of nitrogen heterocycles.At the same time, it was demonstrated that the inhibitory effect of GA derivatives modified with azoles AGAs1-3 on F. graminearum was a synergistic effect of azoles and GA, which was consistent with our expectations.These results indicate that the structure of GA derivatives containing acyl imidazole, acyl pyrazole and imidazolium salts effectively enhances its antifungal activity against F. graminearum.This phenomenon may be attributed to the p-π conjugation between acylimidazole and acylpyrazole heterocyclic groups, which can form a large conjugated system in GA derivatives containing acyl azole structures.In addition, the lone pair electrons of the heteroatom (N) in pyrazole and imidazole increased the density of the electron cloud on the derivatives of GA, which could enhance the interaction between the compounds and its receptor, thereby improving the antifungal effect.In addition, the possible reason for AGAs-3 to have the highest antifungal activity against F. graminearum might be attributed to the quaternary ammonium salt structure itself.
Molecules 2024, 29, x FOR PEER REVIEW 4 of 15 were both zero at concentrations of 0.05-0.4mg/mL.When the concentration was 0.8 mg/mL, the inhibitory rates on the mycelial growth of F. graminearum were 9.93% and 9.55%, respectively.It can be seen that at different concentrations, the inhibitory effect of compounds AGAs1-3 on the mycelial growth of F. graminearum is significantly be er than that of unmodified compound GA, indicating that compared with the unmodified complex GA, the azole-modified GA derivatives AGAs1-3 significantly enhance its antifungal activity against F. graminearum due to the introduction of nitrogen heterocycles.At the same time, it was demonstrated that the inhibitory effect of GA derivatives modified with azoles AGAs1-3 on F. graminearum was a synergistic effect of azoles and GA, which was consistent with our expectations.These results indicate that the structure of GA derivatives containing acyl imidazole, acyl pyrazole and imidazolium salts effectively enhances its antifungal activity against F. graminearum.This phenomenon may be a ributed to the p-π conjugation between acylimidazole and acylpyrazole heterocyclic groups, which can form a large conjugated system in GA derivatives containing acyl azole structures.In addition, the lone pair electrons of the heteroatom (N) in pyrazole and imidazole increased the density of the electron cloud on the derivatives of GA, which could enhance the interaction between the compounds and its receptor, thereby improving the antifungal effect.In addition, the possible reason for AGAs-3 to have the highest antifungal activity against F. graminearum might be a ributed to the quaternary ammonium salt structure itself.Spore germination is a crucial step for filamentous fungi to colonize in new environments, leading to the formation of polarized growth axes.Therefore, the inhibitory activity of five compounds on spore germination was tested.Figure 3B shows the inhibitory rates of five compounds at a concentration of 0.4 mg/mL on the germination of F. graminearum spores.The inhibitory rates of the five compounds on F. graminearum spore germination ranged from 70% to 96%, indicating that they all had a certain protective effect against Fusarium head blight.The inhibitory rate of AGAs-3, the gallic acid derivative imidazolium salt, on F. graminearum spore germination was 95.45%, indicating that the protective effect of AGAs-3 compounds against wheat scab was significantly improved compared to GA.This might be due to the fact that compound AGAs-3 has a quaternary ammonium salt structure and exhibits a positive charge in aqueous solutions, Spore germination is a crucial step for filamentous fungi to colonize in new environments, leading to the formation of polarized growth axes.Therefore, the inhibitory activity of five compounds on spore germination was tested.Figure 3B shows the inhibitory rates of five compounds at a concentration of 0.4 mg/mL on the germination of F. graminearum spores.The inhibitory rates of the five compounds on F. graminearum spore germination ranged from 70% to 96%, indicating that they all had a certain protective effect against Fusarium head blight.The inhibitory rate of AGAs-3, the gallic acid derivative imidazolium salt, on F. graminearum spore germination was 95.45%, indicating that the protective effect of AGAs-3 compounds against wheat scab was significantly improved compared to GA.This might be due to the fact that compound AGAs-3 has a quaternary ammonium salt structure and exhibits a positive charge in aqueous solutions, making it easier to bind to the fungal spore cell membranes.By changing the permeability of cell membranes, making enzymes or proteins denatured, affecting cell metabolism and inhibiting their activity, AGAs-3 achieves antifungal effects [40].
Additionally, the EC 50 values of the five compounds (GA, TMBA, and AGAs1-3) on the growth of F. graminearum mycelium are shown in Table 1, which indicates that the EC 50 of compounds AGAs1-3 is significantly lower than that of GA.Especially for AGAs-3, the EC 50 value is 0.42 mg/mL.In addition, it can be clearly seen from the experimental photos of mycelial growth rate that compounds AGAs1-3 have a significant inhibitory effect on the growth of F. graminearum hyphae in a dose-dependent manner (Figure 4).making it easier to bind to the fungal spore cell membranes.By changing the permeability of cell membranes, making enzymes or proteins denatured, affecting cell metabolism and inhibiting their activity, AGAs-3 achieves antifungal effects [40].Additionally, the EC50 values of the five compounds (GA, TMBA, and AGAs1-3) on the growth of F. graminearum mycelium are shown in Table 1, which indicates that the EC50 of compounds AGAs1-3 is significantly lower than that of GA.Especially for AGAs-3, the EC50 value is 0.42 mg/mL.In addition, it can be clearly seen from the experimental photos of mycelial growth rate that compounds AGAs1-3 have a significant inhibitory effect on the growth of F. graminearum hyphae in a dose-dependent manner (Figure 4).The above research results indicate that unmodified GA exhibits extremely low antifungal activity in inhibiting the growth of F. graminearum hyphae at different concentration levels.However, the introduction of imidazole, pyrazole, and imidazolium salt significantly enhanced the antifungal activity of GA against F. graminearum, indicating that the production of pharmacological groups such as acylimidazole, acylpyrazole, and imidazolium salts in GA derivatives play a crucial inhibitory role on the growth of F. graminearum hyphae.The possible reason for this might be, on the one hand, that the specific chemical structure of GA derivatives AGAs1-3 can affect the electron transfer process which plays an important role in its interaction with fungal cell membrane receptors.On the other hand, different mechanisms such as chemical reactions, biological The above research results indicate that unmodified GA exhibits extremely low antifungal activity in inhibiting the growth of F. graminearum hyphae at different concentration levels.However, the introduction of imidazole, pyrazole, and imidazolium salt significantly enhanced the antifungal activity of GA against F. graminearum, indicating that the production of pharmacological groups such as acylimidazole, acylpyrazole, and imidazolium salts in GA derivatives play a crucial inhibitory role on the growth of F. graminearum hyphae.The possible reason for this might be, on the one hand, that the specific chemical structure of GA derivatives AGAs1-3 can affect the electron transfer process which plays an important role in its interaction with fungal cell membrane receptors.On the other hand, different mechanisms such as chemical reactions, biological effects, and physical barriers when the mycelium interacts with the GA derivatives AGAs1-3, result in potential damage to the mycelium.These mechanisms may individually or in coordination promote antifungal activity against F. graminearum [41].

The Effect of Compound AGAs-3 on Mycelial Morphology
The morphology of F. graminearum mycelium was observed under scanning electron microscopy (SEM) (Figure 5).From the SEM images, we can see that the mycelium of F. graminearum in the blank group grows vigorously, with a good extensibility, uniform thickness, and a relatively plump and smooth surface morphology (Figure 5A).After treatment with compound AGAs-3, the surface morphology of the mycelium of F. graminearum underwent significant collapse, deformation, shrinkage, and varying degrees of distortion (Figure 5B).
Molecules 2024, 29, x FOR PEER REVIEW 6 of 15 effects, and physical barriers when the mycelium interacts with the GA derivatives AGAs1-3, result in potential damage to the mycelium.These mechanisms may individually or in coordination promote antifungal activity against F. graminearum [41].

The Effect of Compound AGAs-3 on Mycelial Morphology
The morphology of F. graminearum mycelium was observed under scanning electron microscopy (SEM) (Figure 5).From the SEM images, we can see that the mycelium of F. graminearum in the blank group grows vigorously, with a good extensibility, uniform thickness, and a relatively plump and smooth surface morphology (Figure 5A).After treatment with compound AGAs-3, the surface morphology of the mycelium of F. graminearum underwent significant collapse, deformation, shrinkage, and varying degrees of distortion (Figure 5B).

Field Efficacy Trials of GA and Its Azole Derivatives against F. graminearum In Vivo
After the ascospores of F. graminearum spread to wheat ears, they germinate and produce hyphae when exposed to water at a certain temperature, invading the tissue of wheat ears and causing disease.The incidence rate and disease index of wheat scab disease under the treatment of GA and its derivatives TMBA and AGAs1-3 in the field test are shown in Table 2. Compared with GA, the azole-modified gallic acid derivatives AGAs-2 and AGAs-3 significantly reduced the disease incidence rate and the disease severity index is much lower than that of the blank group.The preventive effect of compound AGAs-3 is as high as 72.11%, indicating that its antifungal activity is significantly be er than that of unmodified compounds, which is consistent with the results of the spore germination experiment.These results indicate that compounds AGAs-2 and AGAs-3 can serve as potential plant-based fungicides in the field of plant protection.

Field Efficacy Trials of GA and Its Azole Derivatives against F. graminearum In Vivo
After the ascospores of F. graminearum spread to wheat ears, they germinate and produce hyphae when exposed to water at a certain temperature, invading the tissue of wheat ears and causing disease.The incidence rate and disease index of wheat scab disease under the treatment of GA and its derivatives TMBA and AGAs1-3 in the field test are shown in Table 2. Compared with GA, the azole-modified gallic acid derivatives AGAs-2 and AGAs-3 significantly reduced the disease incidence rate and the disease severity index is much lower than that of the blank group.The preventive effect of compound AGAs-3 is as high as 72.11%, indicating that its antifungal activity is significantly better than that of unmodified compounds, which is consistent with the results of the spore germination experiment.These results indicate that compounds AGAs-2 and AGAs-3 can serve as potential plant-based fungicides in the field of plant protection.Figure 6 shows the antifungal activity of GA and its derivatives TMBA and AGAs1-3 against F. graminearum.In field experiments, the antifungal activity of compounds AGAs-2 and AGAs-3 against F. graminearum was significantly better than other compounds, indicating that these two compounds have good antifungal activity in vitro and are a promising antifungal agent for controlling crop fungal diseases.
Figure 6 shows the antifungal activity of GA and its derivatives TMBA and AGA 3 against F. graminearum.In field experiments, the antifungal activity of compoun AGAs-2 and AGAs-3 against F. graminearum was significantly be er than ot compounds, indicating that these two compounds have good antifungal activity in vi and are a promising antifungal agent for controlling crop fungal diseases.Taking the imidazolium salt AGAs-3, which has the highest inhibitory activ against F. graminearum, as an example, the distribution of the HOMO and LUMO orbit of compound AGAs-3 were calculated at the same theoretical level, as shown in Figur It can be observed that the HOMO of AGAs-3 is mainly located on the chlorine atom the molecule, which is determined by the strong electron withdrawing effect of chlorine atom (Figure 7A).The LUMO of AGAs-3 is mainly located in the region wh the imidazole ring and carbonyl group are located (Figure 7B), which are the active cent that play a key role in the interaction between AGAs-3 and fungal cell membra receptors.When it interacts with the cell membrane receptor, the chlorine atom provi electrons to the receptor, while where the imidazole ring and carbonyl group are loca is an onium salt structure, and of which the other side connecting the carbonyl group benzene ring structure.All of these can form a large, delocalized structure, promoting stable flow of electrons from the cell membrane receptor toward the delocalized regi and causing it to bind with the cell membrane receptor to form electron trans complexes, breaking the original structure of the cell membrane receptor, ultimat leading to changes in the regulatory mechanism of the cell membrane receptor a achieving antifungal effects.

Theoretical Calculation
Frontier molecular orbitals are important predictive indicators of molecular polarization and reactivity.Most chemical reactions occur by transferring electrons from the highest occupied molecular orbital (HOMO) of the donor to the lowest unoccupied molecular orbital (LUMO) of the acceptor [42].When drug molecules interact with fungi, they may utilize electron transfer and other interactions between drug molecules and the main components of fungal cell walls, such as polysaccharides, proteins, or lipid receptors, to achieve antifungal effects.
Taking the imidazolium salt AGAs-3, which has the highest inhibitory activity against F. graminearum, as an example, the distribution of the HOMO and LUMO orbitals of compound AGAs-3 were calculated at the same theoretical level, as shown in Figure 6.It can be observed that the HOMO of AGAs-3 is mainly located on the chlorine atom in the molecule, which is determined by the strong electron withdrawing effect of the chlorine atom (Figure 7A).The LUMO of AGAs-3 is mainly located in the region where the imidazole ring and carbonyl group are located (Figure 7B), which are the active centers that play a key role in the interaction between AGAs-3 and fungal cell membrane receptors.When it interacts with the cell membrane receptor, the chlorine atom provides electrons to the receptor, while where the imidazole ring and carbonyl group are located is an onium salt structure, and of which the other side connecting the carbonyl group is a benzene ring structure.All of these can form a large, delocalized structure, promoting the stable flow of electrons from the cell membrane receptor toward the delocalized region, and causing it to bind with the cell membrane receptor to form electron transfer complexes, breaking the original structure of the cell membrane receptor, ultimately leading to changes in the regulatory mechanism of the cell membrane receptor and achieving antifungal effects.Combining the molecular electrostatic potential (MEP) plot can more intuitively illustrate this phenomenon.The MEP plot is related to the total electron density and is commonly used to identify regions that are prone to electrophilic and nucleophilic reactions, as well as hydrogen bonding interactions, and to define regions with local negative and positive charges within the molecule.MEP reflects the electron density and indicates the affinity between drugs and receptors.Figure 7D shows the MEP of AGAs-3, with a blue electron rich region indicating that electrons are at a high energy level and have a large number of electrons, making it easy to abandon them.Due to the strong electron withdrawing effect of Cl atoms, a large amount of negative charges are accumulated in this region.Therefore, when compound AGAs-3 interacts with the protein or enzyme of the receptor, this part mainly provides electrons, forming an electron transfer process, thereby achieving antifungal effects.The imidazole ring and carbonyl structure of compound AGAs-3 are shown in red on the MEP diagram, indicating that the accumulation of a significant amount of positive charge in this region, which mainly receives electrons provided by the receptor when interacting with it.

Materials
Silicone (100-200 mesh/200-300 mesh, Qingdao Gulf Fine Chemical Co., Ltd., Qingdao, China), dimethyl sulfate (AR, Chengdu Huaxia Chemical Reagent Co., Ltd., Chengdu, China), imidazole (AR, Shanghai Darui Fine Chemicals Co., Ltd., Shanghai, China), pyrazole (AR, Shanghai Darui Fine Chemicals Co., Ltd., Shanghai, China), benzyl chloride (AR, Aladdin Reagent Co., Ltd., Shanghai, China), 1-ethyl-(3dimethylaminopropyl) carbodiimide (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) (AR, Tianjin heowns bio-chemical Technology Co., Ltd., Tianjin, China), 1hydroxybenzotriazole (HOBT) (AR, Tianjin heowns bio-chemical Technology Co., Ltd., Tianjin, China), concentrated hydrochloric acid, sodium hydroxide, anhydrous sodium sulfate (Na2SO4), anhydrous ether, anhydrous ethanol, petroleum ether, and ethyl acetate analytical purity agents were purchased from Tianjin Oubokai Chemical Co., Ltd.(Tianjin, China), while dichloromethane (CH2Cl2) and acetonitrile chromatographic purity agents Combining the molecular electrostatic potential (MEP) plot can more intuitively illustrate this phenomenon.The MEP plot is related to the total electron density and is commonly used to identify regions that are prone to electrophilic and nucleophilic reactions, as well as hydrogen bonding interactions, and to define regions with local negative and positive charges within the molecule.MEP reflects the electron density and indicates the affinity between drugs and receptors.Figure 7D shows the MEP of AGAs-3, with a blue electron rich region indicating that electrons are at a high energy level and have a large number of electrons, making it easy to abandon them.Due to the strong electron withdrawing effect of Cl atoms, a large amount of negative charges are accumulated in this region.Therefore, when compound AGAs-3 interacts with the protein or enzyme of the receptor, this part mainly provides electrons, forming an electron transfer process, thereby achieving antifungal effects.The imidazole ring and carbonyl structure of compound AGAs-3 are shown in red on the MEP diagram, indicating that the accumulation of a significant amount of positive charge in this region, which mainly receives electrons provided by the receptor when interacting with it.

Characterization
The structures of all the final products were identified and confirmed by nuclear magnetic resonance spectroscopy (NMR, 600 MHz, Bruker, Germany) and high resolution mass spectroscopy (HR-ESI-MS, LTQ Orbitrap XL, Thermo Fisher, Waltham, MA, USA).The mycelial morphology of F. graminearum was observed with scanning electron microscopy (SEM, SU8010, Hitachi, Tokyo, Japan).

Mycelial Growth Rate Experiment
We mixed 444.4 g of potatoes, 35.2 g of agar, and 35.2 g of sugar in 450 mL of water and heated it to boiling to produce PDA.Then, we divided the PDA into several conical bottles and sealed them at 121 • C for sterilization about 30 min before using.
Three replicates were set for each concentration in this experiment, and PDA was used as a blank control after equal volume sterilization.We weighed 120 mg of GA, added 20 mL of deionized water, and sonicated for 1 h until all GA was dissolved.We mixed it with PDA to prepare toxic culture media with concentrations of 0.05 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.4 mg/mL, and 0.8 mg/mL.Then, we inoculated F. graminearum onto a toxic culture medium and cultured continuously for three days at a temperature of 28 ± 1 • C. All in vitro antifungal test methods for drugs were carried out according to the above procedure.
We used the cross-over method to determine the colony diameter, calculated the average diameter size, and calculated the antibacterial rate of the drug according to Formula (1) [44,45]: where dt is the average colony diameter of the treatment group, and dc is the average colony diameter of the blank control group.

In Vitro Antibacterial Activity Testing of Spore Germination
We made 100 µL spore suspension (3 × 10 −7 spore mL −1 ) and 100 µL solution containing AGAs-3 to prepare a drug spore suspension with a concentration of 0.4 mg/mL.Then, we took 30 µL of the above suspension and placed it on a sterile concave glass slide.After completely dark cultivation at 28 ± 1 • C for 5 h, we measured the spore germination rate and at least 200 spores per treatment.

Methods of Sample Preparation for Scanning Electron Microscopy
The cultured hyphae was fixed in a refrigerator at 4 • C with 2.5% pH = 6.8 glutaraldehyde for 6 h, and then rinsed with 0.1 mol/L pH = 6.8 phosphate buffer solution, followed by gradient dehydration and displacement with different concentrations of ethanol and tert-butanol.Finally, the sample was freeze-dried and sprayed with a layer of 1500 nm thick gold film.

Antifungal Activity of GA and Its Derivatives In Vivo
Field efficacy trials of GA and its azole derivatives (GA, TMBA, AGAs1-3) against wheat scab were conducted at the experimental station of Hebei Normal University of Science and Technology.Wheat seeds (Shaochuan 02-1) were purchased from Hebei Academy of Agricultural and Forestry Sciences.The antifungal activity of compounds GA, TMBA, and AGAs1-3 against F. graminearum was determined using the single-sided small flower inoculation method [46].During the early flowering stage of wheat, spore suspension was injected into a single side small flower of a spike (F.graminearum spore suspension was mixed with compounds GA, TMBA, and AGAs1-3 to form a 0.4 mg/mL inoculum, and sterile water was used as a negative control).We used self-sealing bags to seal wheat ears for three days to maintain humidity and promote disease development.Seven days after 100 spikelets were inoculated with each drug, the incidence rate and disease index of spikelets were counted [47].Seven days after onset, the incidence rate of disease and disease index were visually assessed using a severity level of 0-100%.
Classification method: Level 0, Whole ear disease-free; Level 1, the area of withered panicles accounts for less than 1/4 of the total panicle area; Level 3, the area of withered panicles accounts for 1/4-1/2 of the total panicle area; Level 5, the area of withered panicles accounts for 1/2-3/4 of the total panicle area; Level 7, the area of withered panicles accounts for more than 3/4 of the total panicle area.
We calculated the diseased ear rate according to Formula (3), where D is the diseased panicle rate, in percentage, and N d is the number of diseased ears, N is the total number of surveys.The disease severity index was calculated according to Formula (4), X = ∑ (N i × i)/N × 7 × 100 (4 where i is the severity levels of the condition.

Theoretical Calculation
Quantum chemical calculations were completed using the Gaussian 09 D.01 program [48].Structural optimization was completed using B3LYP [49,50] functional combined with 6-31G (d, p) basis set [51].The molecular surface electrostatic potential map and frontier molecular orbital contour map were rendered using the VMD 1.9.3 program [52], and the VMD rendered files were obtained from Multiwfn 3.8(dev) code [53].

Conclusions
Reasonable use of pesticides is an important measure to prevent and control wheat scab to improve the yield and quality of wheat.This article successfully synthesized three new compounds using natural polyphenol GA as raw material, and identified their structures through NMR and HRMS.The antifungal ability of GA and its derivatives against F. graminearum was measured at different concentrations using F. graminearum as a model strain.The results showed that compared with GA, AGAs1-3 exhibited significant antifungal activity against F. graminearum in a dose-dependent manner.The introduction of imidazolium salt structure can significantly inhibit the growth of F. graminearum with an EC 50 of 0.42 mg/mL and an inhibition rate of 73.5% on the mycelial growth of F. graminearum.When 0.4 mg/mL of AGAs-3 was applied, the control effect of wheat FHB in the field reached 72.11%, significantly reducing the degree of infection of F. graminearum on wheat, demonstrating a good control effect.Therefore, GA derivatives with imidazolium salt structure can provide a theoretical basis for the development of green and environmentally friendly fungicides for plant pathogenic fungi.

Figure 1 .
Figure 1.The structures of imidazole and pyrazole derivatives with various biological activities.Figure 1.The structures of imidazole and pyrazole derivatives with various biological activities.

Figure 1 .
Figure 1.The structures of imidazole and pyrazole derivatives with various biological activities.Figure 1.The structures of imidazole and pyrazole derivatives with various biological activities.

Figure 3 .
Figure 3. Effects of five compounds GA, TMBA, AGAs1-3 on different concentrations at 28 ± 1 °C.The inhibitory effect of F. graminearum mycelium growth (A); and the inhibitory rates of five compounds GA, TMBA, and AGAs1-3 on spore germination at 28 ± 1 °C for 5 h (B).The error bars represent the standard error (N = 3), and different lowercase le ers indicate significant differences between treatments (p < 0.05).

Figure 3 .
Figure 3. Effects of five compounds GA, TMBA, AGAs1-3 on different concentrations at 28 ± 1 • C. The inhibitory effect of F. graminearum mycelium growth (A); and the inhibitory rates of five compounds GA, TMBA, and AGAs1-3 on spore germination at 28 ± 1 • C for 5 h (B).The error bars represent the standard error (N = 3), and different lowercase letters indicate significant differences between treatments (p < 0.05).

Figure 4 .
Figure 4.The antifungal activity of GA and its derivatives against F. graminearum in vitro (cultured continuously at 28 ± 1 °C for 3 days, mg/mL).

Figure 4 .
Figure 4.The antifungal activity of GA and its derivatives against F. graminearum in vitro (cultured continuously at 28 ± 1 • C for 3 days, mg/mL).

Figure 6 .
Figure 6.In vivo antifungal activity against F. graminearum under different treatments (0.4 mg/m 2.5.Theoretical Calculation Frontier molecular orbitals are important predictive indicators of molecu polarization and reactivity.Most chemical reactions occur by transferring electrons fr the highest occupied molecular orbital (HOMO) of the donor to the lowest unoccup molecular orbital (LUMO) of the acceptor [42].When drug molecules interact with fun they may utilize electron transfer and other interactions between drug molecules and main components of fungal cell walls, such as polysaccharides, proteins, or li receptors, to achieve antifungal effects.Taking the imidazolium salt AGAs-3, which has the highest inhibitory activ against F. graminearum, as an example, the distribution of the HOMO and LUMO orbit of compound AGAs-3 were calculated at the same theoretical level, as shown in Figur It can be observed that the HOMO of AGAs-3 is mainly located on the chlorine atom the molecule, which is determined by the strong electron withdrawing effect of chlorine atom (Figure7A).The LUMO of AGAs-3 is mainly located in the region wh the imidazole ring and carbonyl group are located (Figure7B), which are the active cent that play a key role in the interaction between AGAs-3 and fungal cell membra receptors.When it interacts with the cell membrane receptor, the chlorine atom provi electrons to the receptor, while where the imidazole ring and carbonyl group are loca is an onium salt structure, and of which the other side connecting the carbonyl group benzene ring structure.All of these can form a large, delocalized structure, promoting stable flow of electrons from the cell membrane receptor toward the delocalized regi and causing it to bind with the cell membrane receptor to form electron trans complexes, breaking the original structure of the cell membrane receptor, ultimat leading to changes in the regulatory mechanism of the cell membrane receptor a achieving antifungal effects.

Table 1 .
The EC 50 values of compounds GA, TMBA, and AGAs1-3 in inhibiting the mycelial growth of F. graminearum.

Table 1 .
The EC50 values of compounds GA, TMBA, and AGAs1-3 in inhibiting the mycelial growth of F. graminearum.

Table 2 .
Field control effects of compounds on F. graminearum.

Table 2 .
Field control effects of compounds on F. graminearum.